There are 30 recorded epizootics of H5 or H7 high pathogenicity avian influenza (HPAI) from 1959 to early 2012. The largest of these epizootics, affecting more birds and countries than the other 29 epizootics combined, has been the H5N1 HPAI, which began in Guangdong China in 1996, and has killed or resulted in culling of over 250 million poultry and/or wild birds in 63 countries. Most countries have used stamping-out programs in poultry to eradicate H5N1 HPAI. However, 15 affected countries have utilized vaccination as a part of the control strategy. Greater than 113 billion doses were used from 2002 to 2010. Five countries have utilized nationwide routine vaccination programs, which account for 99% of vaccine used: 1) China (90.9%), 2) Egypt (4.6%), 3) Indonesia (2.3%), 4) Vietnam (1.4%), and 5) Hong Kong Special Administrative Region (<0.01%). Mongolia, Kazakhstan, France, The Netherlands, Cote d'Ivoire, Sudan, North Korea, Israel, Russia, and Pakistan used <1% of the avian influenza (AI) vaccine, and the AI vaccine was targeted to either preventive or emergency vaccination programs. Inactivated AI vaccines have accounted for 95.5% of vaccine used, and live recombinant virus vaccines have accounted for 4.5% of vaccine used. The latter are primarily recombinant Newcastle disease vectored vaccine with H5 influenza gene insert. China, Indonesia, Egypt, and Vietnam implemented vaccination after H5N1 HPAI became enzootic in domestic poultry. Bangladesh and eastern India have enzootic H5N1 HPAI and have not used vaccination in their control programs. Clinical disease and mortality have been prevented in chickens, human cases have been reduced, and rural livelihoods and food security have been maintained by using vaccines during HPAI outbreaks. However, field outbreaks have occurred in vaccinating countries, primarily because of inadequate coverage in the target species, but vaccine failures have occurred following antigenic drift in field viruses within China, Egypt, Indonesia, Hong Kong, and Vietnam. The primary strategy for HPAI and H5/H7 low pathogenicity notifiable avian influenza control will continue to be immediate eradication using a four-component strategy: 1) education, 2) biosecurity, 3) rapid diagnostics and surveillance, and 4) elimination of infected poultry. Under some circumstances, vaccination can be added as an additional tool within a wider control strategy when immediate eradication is not feasible, which will maintain livelihoods and food security, and control clinical disease until a primary strategy can be developed and implemented to achieve eradication.

Three broad factors, occurring concurrently, prevent elimination of highly pathogenic avian influenza caused by viruses of the H5N1 subtype (H5N1 HPAI) in countries and subregions where infection has remained endemic. These factors are the nature of the poultry sector, the quality of veterinary and animal production services (both public and private) serving the poultry industry, and the extent and level of commitment at all levels to virus elimination. Most of these countries have developed and adopted programs for progressive control of H5N1 HPAI, focused on the local factors hindering elimination of the viruses. Based on the rate of implementation of these measures over the last 5 to 7 yr (during which time there has been unprecedented financial and technical support from the international donor community), it is not expected that global eradication of H5N1 HPAI viruses can be achieved within the next 10 yr. If the “classical” approach to control, based around early case detection and culling, were adopted in a zone containing millions of free-running ducks, the work load required to complete even the first round of testing would exceed existing capacity. There would be no guarantees of sustained success locally, especially if the viruses are not eradicated regionally.

Vaccination for both low pathogenicity avian influenza and highly pathogenic avian influenza is commonly used by countries that have become endemic for avian influenza virus, but stamping-out policies are still common for countries with recently introduced disease. Stamping-out policies of euthanatizing infected and at-risk flocks has been an effective control tool, but it comes at a high social and economic cost. Efforts to identify alternative ways to respond to outbreaks without widespread stamping out has become a goal for organizations like the World Organisation for Animal Health. A major issue with vaccination for avian influenza is trade considerations because countries that vaccinate are often considered to be endemic for the disease and they typically lose their export markets. Primarily as a tool to promote trade, the concept of DIVA (differentiate infected from vaccinated animals) has been considered for avian influenza, but the goal for trade is to differentiate vaccinated and not-infected from vaccinated and infected animals because trading partners are unwilling to accept infected birds. Several different strategies have been investigated for a DIVA strategy, but each has advantages and disadvantages. A review of current knowledge on the research and implementation of the DIVA strategy will be discussed with possible ways to implement this strategy in the field. The increased desire for a workable DIVA strategy may lead to one of these ideas moving from the experimental to the practical.

All reports of avian influenza virus infections in poultry and isolations from wild bird species in Canada, the United States, and Mexico between 2009 and 2011 involved low pathogenic avian influenza. All three countries reported outbreaks of low pathogenic notifiable avian influenza in poultry during this period. The reports involved outbreaks of H5N2 among commercial turkeys in Canada in 2009 and 2010; outbreaks of H5N3 in turkeys in 2009, H5N2 in chickens in 2010, H7N3 in turkeys in 2011, and H7N9 in chickens, turkeys, geese, and guinea fowl in 2011 in the United States; and multiple outbreaks of H5N2 in chickens in Mexico in 2009, 2010, and 2011. Outbreaks of pandemic H1N1 infections in turkey breeder flocks were reported in Canada in 2009 and in the United States in 2010. Active surveillance of live bird markets in the United States led to the detection of H2, H3, H4, H5, H6, and H10 subtypes. Despite the fact that wild bird surveillance programs underwent contraction during this period in both Canada and the United States, H5 and H7 subtypes were still detected.

Widespread prevalence of avian influenza H9N2 subtype in the Middle East region and its detection in Egypt in quail in early summer 2011 added another risk factor to the Egyptian poultry industry in addition to highly pathogenic H5N1 subtype. This situation increases the need for further surveillance and investigation of H9N2 viruses in commercial and household chickens. This work describes detection and genetic characterization of recently isolated H9N2 viruses from chicken flocks. Parallel detection and genetic characterization of H5N1 viruses from infections in poultry has also been done to compare the prevalence of the two subtypes in close geographic locations in Egypt. Phylogenetic analysis of the HA gene showed that the Egyptian isolates of H9N2 were grouped together within the quail/Hong Kong/G1/97-like lineage, similar to the circulating viruses in the Middle East, with very close phylogeny to the Israeli viruses. The prevalence of H5N1 viruses from cases recorded in poultry in the nearby areas revealed a marked decrease in disease incidence in commercial broilers but an increased incidence in household birds. The genetic characterization of the H5N1 viruses indicated predominance of the classic 2.2.1 subclade, with evolution of new viruses and no detection for the variant 2.2.1.1 subclade. The cocirculation of the two subtypes, H5N1 and H9N2, of avian influenza may affect the limit of spread and the epizootiologic pattern of the infections for both subtypes, especially when different vaccination and biosecurity approaches are applied in the field level.

The objective of this study was to determine the prevalence of avian influenza viruses (AIV) in bobwhite quail (Colinus virginianus) populations from the rolling plains of Texas, U. S. A. A total of 1320 swab samples (652 tracheal swabs and 668 cloacal swabs) and 44 serum samples were collected from wild-captured or hunter-harvested bobwhite quail from November 2009 to April 2011 at the Rolling Planes Quail Research Ranch, Fisher County, Texas, U. S. A. The presence of AIV in the swabs was determined by real-time reverse-transcription–PCR (rRT-PCR) and all samples positive or suspicious by rRT-PCR were further processed for virus isolation in embryonated chicken eggs. A total of 18 (1.4%) swab samples tested positive for AIV by rRT-PCR (cycle threshold [Ct] values <35): 13 cloacal swabs (1.9%) and 5 tracheal swabs (0.8%). In addition, 100 (7.6%) swab samples were considered suspicious (Ct values 35.1–40): 69 cloacal swabs (10.3%) and 31 tracheal swabs (4.7%). No virus was isolated from any of the rRT-PCR–positive or suspicious samples tested. Additionally, 44 serum samples were screened for AIV antibodies and were negative. The results presented here indicate low prevalence of AIV in wild populations of bobwhite quail.

Five outbreaks of H5N1 highly pathogenic avian influenza (HPAI) have been diagnosed in domestic poultry and wild birds in Cambodia from January to November of 2011. Of the five outbreaks, one occurred in a village backyard flock in Kandal province in January; two occurred in native Cambodian chickens and ducks in Banteay Meanchey province in July and August, respectively; one was seen in wild birds in Phnom Tamao Zoo in Kandal Province in July; and one outbreak occurred in commercial broilers at Opong Moan in Battambang province in northwestern Cambodia in early November. Clinically, HPAI-infected broilers and native chickens showed sudden death, severe depression, ruffled feathers, edema of heads and necks, swollen and cyanotic combs and wattles, and swollen and congested conjunctiva, with occasional hemorrhage, paralysis, and other neurologic signs. In ducks, significantly swollen sinuses and eyes, cloudy corneas, difficulty standing, or paralysis were commonly seen. Some affected ducks showed sudden death without obvious clinical symptoms. Necropsy lesions showed congestion and necrotic debris within sinuses and severe hemorrhages in gizzards, livers, and lungs in both affected native chickens and ducks during the new outbreaks in 2011. All five outbreaks were diagnosed as H5N1 HPAI by virus isolation and real-time reverse transcription–PCR tests. Once a backyard flock in a village or a poultry farm was diagnosed as positive for H5N1 HPAI; the whole village backyard poultry and all farm flocks were culled immediately by Cambodian provincial and central authorities as per the strategies adopted for the control of HPAI.

The third outbreak of highly pathogenic avian influenza (HPAI) H5N2 in less than seven years affected ostriches of South Africa's Western Cape during 2011. Twenty farms tested PCR positive for the presence of HPAI H5N2 between March and November 2011. Three HPAI H5N2 (AI2114, AI2214, AI2512) and 1 H1N2 (AI2887) viruses were isolated during this period, but H6N2 and H1N2 infections of ostriches were also confirmed by PCR. HPAI H5N2 isolate AI2114 produced an intravenous pathogenicity index (IVPI) score of 1.37 in chickens whereas isolate AI2214 produced an IVPI score of 0.8. The former virus had an additional, predicted N-linked glycosylation site at position 88 of the hemagglutinin protein as well as an E627K mutation in the PB2 protein that was lacking from AI2214. Four variations at HA₀ were detected in the PCR-positive cases. Phylogenetically, the branching order of outbreak strains indicated a lack of reassortment between outbreak strains that implied a single outbreak source and a wild duck origin for the progenitor outbreak strain. The 2011 outbreak strains had no genetic relationships to the previous 2004 and 2006 HPAI H5N2 outbreak viruses. Molecular clock analysis based on the N2 neuraminidase genes estimated a recent common ancestor for the outbreak tentatively dated at September 2010. Deep sequencing results of 16 clinical PCR-positive samples yielded data in the range of 573 to 12,590 base pairs (bp), with an average of 4468 bp of total genomic sequence recovered per sample. This data was used to confirm the lack of reassortment and to assign samples into one of two epidemiologic groups to support epidemiologic tracing of the spread of the outbreak. One farm (no. 142), thought to have played a major epidemiologic role in the outbreak, was confirmed by deep sequencing to contain a mix of both epidemiologic virus groups.

Surveillance, comprised of sampling and testing, of low pathogenic avian influenza virus (LPAIV) in a live bird market (LBM) may enable the detection of the virus, reducing its spread within the market to humans and birds and to other markets within the LBM system. In addition, detection of infected birds would also reduce the probability of reassortment and possible change from a LPAIV to a highly pathogenic avian influenza virus, which would have a devastating impact on the economy, trade, and society. In this paper we present results from a computer simulation model based on previously collected survey and experimental transmission data. Once we validated the model with experimental transmission data, we applied it to address some of the questions that need to be answered in order to create an efficient surveillance system in an LBM. We have identified effective sampling times, patterns, and sizes that would enhance the probability of an early detection of LPAIV if present and minimize the associated labor and cost. The model may be modified to evaluate different sized and structured LBMs. It also provides the basis to evaluate an entire LBM system for the United States or other countries.

When an avian influenza or virulent Newcastle disease outbreak occurs within commercial poultry, key steps involved in managing a fast-moving poultry disease can include: education; biosecurity; diagnostics and surveillance; quarantine; elimination of infected poultry through depopulation or culling, disposal, and disinfection; and decreasing host susceptibility. Available mass emergency depopulation procedures include whole-house gassing, partial-house gassing, containerized gassing, and water-based foam. To evaluate potential depopulation methods, it is often necessary to determine the time to the loss of consciousness (LOC) in poultry. Many current approaches to evaluating LOC are qualitative and require visual observation of the birds. This study outlines an electroencephalogram (EEG) frequency domain–based approach for determining the point at which a bird loses consciousness. In this study, commercial broilers were used to develop the methodology, and the methodology was validated with layer hens. In total, 42 data sets from 13 broilers aged 5–10 wk and 12 data sets from four spent hens (age greater than 1 yr) were collected and analyzed. A wireless EEG transmitter was surgically implanted, and each bird was monitored during individual treatment with isoflurane anesthesia. EEG data were evaluated using a frequency-based approach. The alpha /delta (A/D, alpha: 8–12 Hz, delta: 0.5–4 Hz) ratio and loss of posture (LOP) were used to determine the point at which the birds became unconscious. Unconsciousness, regardless of the method of induction, causes suppression in alpha and a rise in the delta frequency component, and this change is used to determine unconsciousness. There was no statistically significant difference between time to unconsciousness as measured by A/D ratio or LOP, and the A/D values were correlated at the times of unconsciousness. The correlation between LOP and A/D ratio indicates that the methodology is appropriate for determining unconsciousness. The A/D ratio approach is suitable for monitoring during anesthesia, during depopulation, and in situations where birds cannot be readily viewed.

When an avian influenza or virulent Newcastle disease outbreak occurs within commercial poultry, a large number of birds that are infected or suspected of infection must be destroyed on site to prevent the rapid spread of disease. The choice of mass emergency depopulation procedures is limited, and all options have limitations. Water-based foam mass emergency depopulation of poultry was developed in 2006 and conditionally approved by the U.S. Department of Agriculture and American Veterinary Medical Association. Water-based foam causes mechanical hypoxia and can be used for broilers, layers, turkeys, and ducks. The time to physiologic states was evaluated for broilers, layer hens, turkeys, and ducks, comparing water-based foam and CO₂ gas using electroencephalogram (unconsciousness and brain death), electrocardiogram (altered terminal cardiac activity), and accelerometer (motion cessation). In broilers, turkeys, and layer hens, water-based foam results in equivalent times to unconsciousness, terminal convulsions, and altered terminal cardiac activity. With Pekin ducks, however, CO₂ gas resulted in shorter times to key physiologic states, in particular unconsciousness, altered terminal cardiac activity, motion cessation, and brain death.

Emergency response during a highly pathogenic avian influenza (HPAI) outbreak may involve quarantine and movement controls for poultry products such as eggs. However, such disease control measures may disrupt business continuity and impact food security, since egg production facilities often do not have sufficient capacity to store eggs for prolonged periods. We propose the incorporation of a holding time before egg movement in conjunction with targeted active surveillance as a novel approach to move eggs from flocks within a control area with a low likelihood of them being contaminated with HPAI virus. Holding time reduces the likelihood of HPAI-contaminated eggs being moved from a farm before HPAI infection is detected in the flock. We used a stochastic disease transmission model to estimate the HPAI disease prevalence, disease mortality, and fraction of internally contaminated eggs at various time points postinfection of a commercial table-egg layer flock. The transmission model results were then used in a simulation model of a targeted matrix gene real-time reverse transcriptase (RRT)-PCR testing based surveillance protocol to estimate the time to detection and the number of contaminated eggs moved under different holding times. Our simulation results indicate a significant reduction in the number of internally contaminated eggs moved from an HPAI-infected undetected flock with each additional day of holding time. Incorporation of a holding time and the use of targeted surveillance have been adopted by the U.S. Department of Agriculture in their Draft Secure Egg Supply Plan for movement of egg industry products during an HPAI outbreak.

Early detection of highly pathogenic avian influenza (HPAI) infection in commercial poultry flocks is a critical component of outbreak control. Reducing the time to detect HPAI infection can reduce the risk of disease transmission to other flocks. The timeliness of different types of detection triggers could be dependent on clinical signs that are first observed in a flock, signs that might vary due to HPAI virus strain characteristics. We developed a stochastic disease transmission model to evaluate how transmission characteristics of various HPAI strains might effect the relative importance of increased mortality, drop in egg production, or daily real-time reverse transcriptase (RRT)-PCR testing, toward detecting HPAI infection in a commercial table-egg layer flock. On average, daily RRT-PCR testing resulted in the shortest time to detection (from 3.5 to 6.1 days) depending on the HPAI virus strain and was less variable over a range of transmission parameters compared with other triggers evaluated. Our results indicate that a trigger to detect a drop in egg production would be useful for HPAI virus strains with long infectious periods (6–8 days) and including an egg-drop detection trigger in emergency response plans would lead to earlier and consistent reporting in some cases. We discuss implications for outbreak control and risk of HPAI spread attributed to different HPAI strain characteristics where an increase in mortality or a drop in egg production or both would be among the first clinical signs observed in an infected flock.

In countries where avian influenza has become endemic, early vaccination of layer pullets or broilers with classical inactivated vaccines at the hatchery is no longer an option because of interference with passive immunity indirectly induced by the necessary vaccination of the breeders. On the other hand, injection of thousands of chicks from 7 to 10 days old on farms has been determined to be unreliable and, therefore, poorly efficacious. For these reasons, interest has arisen regarding a newly developed live recombinant vector vaccine based on a turkey herpesvirus (HVT) expressing the H5 gene from a clade 2.2 H5N1 highly pathogenic avian influenza virus (HPAIV) strain (rHVT-H5), which in theory is capable of breakthrough passive immunity to both the vector (HVT) and the insert (H5) and is consequently applicable at the hatchery. The objectives of this trial were to evaluate the impact of maternally derived antibodies (MDAs) specific to H5N1 on the immunity and the efficacy (protection and virus shedding) of different vaccination programs including rHVT-H5 and inactivated H5N1 and H5N2 vaccines applied alone or in combination. Therefore, broilers carrying MDAs against both HVT and Asian H5N1 HPAIV were vaccinated on the first day of age with rHVT-H5, with or without boosting vaccination by an inactivated vaccine after 10 days. The different groups were challenged with two antigenically highly divergent Egyptian clade 2.2.1 H5N1 HPAIVs at 4 wk of age. Protection against challenge was compared with unvaccinated birds or vaccinated birds without MDAs. Between 70% and 90% clinical protection could be observed in the vaccinated groups possessing MDAs, indicating no or very low interference of MDAs with vaccination. Results regarding clinical protection, humoral, cell-mediated, and mucosal immunity, as well as re-excretion of challenge virus are presented and discussed.

The swift evolution rate of avian influenza (AI) H5N1 virus demands constant efforts to update inactivated vaccines to match antigenically with the emerging new field virus strains. Recently, a recombinant turkey herpesvirus (rHVT)-AI vaccine, rHVT-H5, expressing the HA gene of a highly pathogenic avian influenza (HPAI) H5N1 clade 2.2 A/Swan/Hungary/499/2006 strain inserted into FC-126 strain of HVT vector, has been developed to combat current threats in poultry industry. Here, we present the results of two trials where rHVT-H5 was tested alone or in combination with inactivated H5N1 vaccines (the latter vaccines contained antigens produced by using a clade 2.1.3 HPAI H5N1 virus [A/Ck/WestJava-Nagrak/2007] in the first trial or mixture of antigen produced by strain A/Ck/WestJava-Nagrak/2007 and A/Ck/Banten-Tangerang/2010 [bivalent vaccine] for second trial) in broiler chickens (Gallus gallus domesticus) carrying maternally derived antibodies to H5N1 and then challenged with Indonesian HPAI H5N1 field isolates. The effectiveness of vaccination was evaluated on the basis of clinical protection (morbidity and mortality) and measurement of virus shedding after challenge. Immune response to vaccination was followed by serology. In the first experiment, chickens were vaccinated at the day of hatch with rHVT-H5 alone (Group 1) or combined with inactivated vaccine at day old (Group 2) or at 10 days of age (Group 3). The chickens along with nonvaccinated hatch-mates were challenged at 28 days of age with the HPAI H5N1 field isolate clade 2.1.3 A/Chicken/WestJava-Subang/29/2007. Eighty, 100%, and 80% clinical protection was recorded in Group 1, 2, and 3, respectively. A similar experiment was performed a second time, but the chicks in Group 3 received the inactivated vaccine earlier, at 7 days of age. Challenge was performed at 28 days of age using a different H5N1 isolate, clade 2.1.3 A/Ck/Purwakarta-Cilingga/142/10. Clinical protection achieved in the second trial was 95%, 75%, and 90% in Group 1, 2, and 3, respectively. Shedding of challenge virus was significantly lower in the vaccinated groups compared with controls in both experiments. Vaccinated birds developed hemagglutination inhibition antibody response to H5N1 by the time of challenge. These experiments confirmed that the rHVT-H5 vaccine applied alone or in combination with inactivated H5N1 vaccines could provide high level (>80%) clinical protection against divergent HPAI H5N1 field isolates after single immunization by 4 wk of age and a significant reduction in the excretion of challenge virus.

For the past decade, several recombinant Newcastle disease viruses (rNDV) have been used as a vector to express native or modified avian influenza (AI) hemagglutinins (HA) in order to give preventive protection against highly pathogenic avian influenza (HPAI) H5N1 viruses. Obtained protections were dependent on the age of the chickens, on the constructs and, in particular, on the homology between the HA that was inserted and the challenge strains. The objective of this study was to investigate the vaccine efficacy of a recombinant NDV La Sota-vectored vaccine expressing an Asian clade 1 H5 ectodomain (rNDV-H5) vaccine expressing a modified H5 ectodomain from an HPAI clade 1 H5N1 isolate as vaccine for 1-day-old specific-pathogen-free chickens. The inoculation route (oculonasal vs. drinking water), the dose-effect, and the protective range of this rNDV-H5 vaccine were studied. Both routes of vaccination induced an H5 serologic response and afforded a high degree of clinical protection against an Asian clade 1 HPAI H5N1 (AsH5N1) challenge without a significant difference between inoculation routes. A clear dose-effect could be demonstrated. Furthermore, when evaluating the protective range against antigenically divergent descendants of the Asian clade 1 HPAI H5N1 lineage, namely two Egyptian clade 2.2.1 H5N1 strains, the vaccine efficacy was less satisfactory. The rNDV-H5 vaccine provided good clinical protection and reduced viral shedding against Egyptian 2007 challenge but was unable to provide a similar protection against the more antigenically divergent Egyptian 2008 strain.

Some H5N1 avian influenza viruses (AIVs) are lethal to quail; however, the use of inactivated vaccines in these birds is largely restricted because of side effects caused by oil adjuvants. Here we evaluated the protective efficacy of a DNA vaccine against lethal challenge with H5N1 highly pathogenic avian influenza virus (HPAIV) in quail. Groups of ten 3-wk-old quail were intramuscularly inoculated three times at 3-wk intervals with 10, 15, 30, or 60 µg, respectively, of plasmid pCAGGoptiHA, which expresses a codon-optimized hemagglutinin gene of the H5N1 virus A/goose/Guangdong/1/96 (GS/GD/96). The control group was inoculated with phosphate-buffered saline. Hemagglutination-inhibition (HI) antibodies were monitored every week after the primary vaccination. The quail were challenged intranasally with 10⁵ EID₅₀ of heterologous HPAIV A/duck/Fujian/31/2007 (DK/FJ/31) (H5N1) 2 wk after the third inoculation. Oropharyngeal and cloacal swab specimens were collected 3, 5, and 7 days after inoculation, and quail were observed daily for disease signs and death for 2 wk. The quail showed no side effects after the plasmid inoculation, and HI antibodies were detectable 1wk after the second vaccination in all groups and increased sharply after the third inoculation. All quail in the PBS-inoculated group and 20% of the birds in the 10 µg plasmid-inoculated group died after the lethal H5N1 virus challenge; however, birds in the 15, 30, and 60 µg plasmid-inoculated groups were completely protected. These results indicate that this DNA vaccine holds promise for use in quail to protect against H5N1 AIV.

Ducks play an important role in the epidemiology of avian influenza, and there is a need for new avian influenza vaccines that are suitable for mass vaccination in ducks. The immune responses as well as highly pathogenic avian influenza (HPAI) H5N1 protection induced by a Newcastle disease virus (NDV) vector expressing an H5N1 hemagglutinin (rNDV-H5) were investigated in mule ducks, a hybrid between Muscovy (Cairina moschata domesticus) males and Pekin (Anas platyrhynchos domesticus) females. Immunological tools to measure NDV and H5-specific serum antibody, mucosal, and cell-mediated immune (CMI) responses in ducks have been validated after infection with the vector NDV and an H5N1 low pathogenic avian influenza virus. The effect of maternally-derived antibodies (MDAs) to NDV on the humoral and CMI responses after NDV-H5 vaccination was also investigated. Our results showed the rNDV-H5 vaccine elicits satisfactory humoral and cellular responses in 11-day-old ducks correlating with a complete clinical and virological protection against the H5N1 strain. However, vaccination with rNDV-H5 in the presence of NDV MDA induced lower NDV-specific serum antibody, mucosal, and CMI responses than in ducks with no MDA, while interestingly the H5-specific serum antibody and duodenal IgY response were higher in ducks with NDV MDA. To our knowledge, this is the first report of the use of an NDV vector in ducks and of an HPAI H5N1 challenge in mule ducks, which appeared to be as resistant as Pekin ducks.

Wild birds that reside in aquatic environments are the major reservoir of avian influenza viruses (AIVs). Since this reservoir of AIVs forms a constant threat for poultry, many countries have engaged in AIV surveillance. More and more commercial enzyme-linked immunosorbent assays (ELISA) are available for serologic surveillance, but these tests are often developed and validated for use in domestic poultry. However, for a correct interpretation of ELISA test results from wild bird sera, more information is needed. In the present study, four ELISA test kits (ID-Vet IDScreen®, IDEXX FlockChek™ AI MultiS-Screen Ab Test Kit, Synbiotics FluDETECT™BE, and BioChek AIMSp) were compared for the serologic analysis of 172 serum samples from mallard, mute swan, and Canada goose. Samples were selected based on ID-Vet IDScreen results to obtain an approximately equal number of positive and negative samples. In addition, 92 serum samples from experimentally infected specific-pathogen-free (SPF) chickens and Pekin ducks were included in the tests for validation purposes. Cohen's kappa statistics and Spearman correlation coefficients were calculated for each combination of two tests and for each bird species. Test agreement for mallard sera varied from poor to moderate, while test results for Canada goose and swan sera agreed from fair to almost perfect. The best agreement was obtained with sera from experimentally infected SPF chickens and Pekin ducks. This study shows that some care must be taken before using nucleoprotein ELISAs for the testing of sera from wild birds and that more reliable validation studies should be considered before their use in the serologic surveillance of wild birds.

Virologic monitoring of avian influenza viruses (AIV) mainly relies on the collection of oropharyngeal, cloacal, or fecal swab samples. The quality of swab samples, therefore, contributes to limitations of the informative value of such monitoring, but the cost of sampling has a great impact on the feasibility of wild bird monitoring studies or poultry surveillance programs. Here, the effect of different swab material and storage conditions on quality and quantity of AIV RNA detection in swab samples by real-time reverse-transcription quantitative PCR has been studied. Two commercial swab products, a rayon-tipped and a flocked nylon type, were compared. Similar suitability of the two swab types, despite a huge price difference, was observed. Superior results by using both types of swab were gained provided that 1) swabs stayed immersed overnight in an appropriate viral transport medium (VTM), or that 2) swabs were vigorously shaken in VTM for at least 1 min and up to 1 hr to release as much trapped virus material as possible. Degradation of RNA over a period of 2 wk for virus-containing samples is negligible when using constant storage conditions at 4 C or 20 C; temperature shifts proved to be more harmful.

During an active wild bird survey conducted in Belgium from 2007 to 2011, two low pathogenic avian influenza (LPAI) H7 viruses were isolated from wild birds: an H7N1 virus from a common shelduck (Tadorna tadorna) and an H7N7 virus from a Canada goose (Branta canadensis). The H7 sequence analyses and intravenous pathogenicity indices indicated that they were both low pathogenic isolates and genetically related to other recent European H7 LPAIs isolated from wild birds. Interestingly, the two isolates showed different replication profiles in specific-pathogen-free (SPF) chickens, but poultry can be at risk from both. Indeed, the H7N1 isolated from the common shelduck had the ability to infect and to replicate efficiently in SPF chickens as indicated by high oropharyngeal and cloacal excretions compatible with efficient transmission as well as strong immune responses. On the other hand, the H7N7 isolated from the Canada goose presented a lower replication profile because the inoculated chickens excreted less virus, mostly via the oropharyngeal route, and only three chickens seroconverted. None of the chickens showed clinical signs during the entire infection. Our study using an SPF chicken model underlines that the mechanisms of adaptation of LPAIs in poultry remain unpredictable and are still poorly understood but it represents a powerful tool to gain a better evaluation of the risks of LPAI circulation in poultry.

In 2011, over 35,000 ostriches were slaughtered in the Oudtshoorn district of the Western Cape province of South Africa following the diagnosis of highly pathogenic avian influenza virus H5N2. We describe the pathology and virus distribution via immunohistochemistry in juvenile birds that died rapidly in this outbreak after showing signs of depression and weakness. Associated sialic acid (SA) receptor distribution in uninfected birds is also described. At necropsy, enlarged spleens, swollen livers, and generalized congestion were noted. Birds not succumbing to acute influenza infection often became cachectic with serous atrophy of fat, airsacculitis, and secondary infections. Necrotizing hepatitis, splenitis, and airsacculitis were prominent histopathologic findings. Virus was detected via immunohistochemistry in abundance in the liver and spleen but also in the air sac and gastrointestinal tract. Infected cells included epithelium, endothelium, macrophages, circulating leukocytes, and smooth muscle of a variety of organs and vessel walls. Analysis of SA receptor distribution in uninfected juvenile ostriches via lectin binding showed abundant expression of SAα2,3Gal (avian type) and little or no expression of SAα2,6Gal (human type) in the gastrointestinal and respiratory tracts, as well as leukocytes in the spleen and endothelial cells in all organs, which correlated with H5N2 antigen distribution in these tissues.

Gulls are widely recognized reservoirs for low pathogenic avian influenza (LPAI) viruses; however, the subtypes maintained in these populations and/or the transmission mechanisms involved are poorly understood. Although, a wide diversity of influenza viruses have been isolated from gulls, two hemagglutinin subtypes (H13 and H16) are rarely detected in other avian groups, and existing surveillance data suggests they are maintained almost exclusively within gull populations. In order to evaluate the host range of these gull-adapted influenza subtypes and to characterize viral infection in the gull host, we conducted a series of challenge experiments, with multiple North American strains of H13 LPAI virus in ring-billed gulls (Larus delawarensis), mallards (Anas platyrhynchos), chickens (Gallus domesticus), and turkeys (Meleagris gallopavo). The susceptibility to H13 LPAI viruses varied between species and viral strain. Gulls were highly susceptible to H13 LPAI virus infection and excreted virus via the oropharynx and cloaca for several days. The quantity and duration of shedding was similar between the two routes. Turkeys and ducks were resistant to infection with most strains of H13 LPAI virus, but low numbers of inoculated birds were infected after challenge with specific viral strains. Chickens were refractory to infection with all strains of H13 LPAI virus they were challenged with. The experimental results presented herein are consistent with existing surveillance data on H13 LPAI viruses in birds, and indicate that influenza viruses of the H13 subtype are strongly host-adapted to gulls, but rare spill-over into aberrant hosts (i.e., turkeys and ducks) can occur.

Mallards are important natural hosts involved in the epidemiology of low pathogenic avian influenza viruses (LPAIVs). LPAIVs are mainly transmitted by a fecal-oral route and are excreted in high concentrations in the feces. We investigated the pathology, viral antigen distribution, and the expression of α2,3 sialic acid (SA) influenza virus receptors in mallards after intranasal inoculation with A/Mallard/MN/199106/99 (H3N8) or A/Mallard/MN/355779/00 (H5N2). Gross lesions were not observed. Avian influenza virus (AIV) nucleoprotein (NP) antigen was detected in rare epithelial cells of the larynx and trachea only at 1-day postinoculation (dpi) in the birds infected with H3N8 LPAIV, but infection with either virus was associated with lymphocytic tracheitis and laryngitis on 1 and 2 dpi. AIV NP antigen was detected in enterocytes of the lower intestine from 1 to 4 dpi and in epithelial cells of the bursa of Fabricius from 2 to 3 dpi in birds infected with either virus. Oropharyngeal and cloacal viral shedding was detected from 1 dpi, with higher cloacal viral shedding detected at 2 and 3 dpi with both viruses. Mallards abundantly expressed α2,3 sialic acid receptors in epithelial cells of the respiratory tract, lower intestine, and bursa of Fabricius. Some infected birds had decreased α2,3 sialic acid expression in epithelial cells of the bursa of Fabricius and in enterocytes of the ceca and colon. In conclusion, the main sites of LPAIV replication in mallards are the enterocytes of the lower intestinal tract and epithelial cells of the bursa of Fabricius in the first days after infection, when these birds are shedding AIV in high titers in the feces.

We studied the effect of different routes of inoculation on the infectivity and duration of viral shedding in mallards (Anas platyrhynchos) infected with wild bird-origin low pathogenic avian influenza viruses (LPAIVs). One-month-old mallards were inoculated with 10⁶ median embryo infectious doses of either A/mallard/MN/199106/99 (H3N8) or A/mallard/MN/355779/00 (H5N2) via 1 of 5 different routes: intranasal (IN), intratracheal (IT), intraocular (IO), intracloacal (IC), or intra-ingluvial (II). Birds in all routes of inoculation groups became infected with LPAIV as detected by virus isolation, real time reverse transcription polymerase chain reaction, and serology. Mallards in different route of inoculation groups had similar viral shedding through oropharynx and cloaca from 1 day postinoculation (dpi). The peak of oropharyngeal (OP) viral shedding was reached between 2 and 3 dpi in all routes of inoculation groups infected with either virus. The peak of cloacal (CL) viral excretion was reached between 2 and 3 dpi in all routes of inoculation groups infected with H3N8 LPAIV and in the IO-, IC-, and II-inoculated groups infected with H5N2 LPAIV, with a delayed and shorter peak for the IN- and IT-inoculated groups. The birds inoculated via the II route had more productive OP and CL viral shedding after infection with either LPAIV, as evidenced by higher number of swabs testing positive over the study period. In conclusion, mallards can be infected with LPAIV by various routes of inoculation, and this corroborates their high susceptibility to infection by these viruses.

This study presents a method for evaluation of surveillance for avian influenza (AI) in wild birds and compares surveillance activities before and after changes in surveillance strategy in Great Britain (GB). In October 2008 the AI Wild Bird Surveillance (AIWBS) system in GB was modified to focus on passive surveillance (birds found dead), including those found during warden patrols of wetlands and wildlife reserves, with less emphasis on public reporting of birds found dead. The number of birds sampled by active surveillance (birds live-trapped or shot) was also reduced. In the present study the impact of these changes was investigated by comparing the 12 mo prior to October 2008 with the subsequent 12 mo. Four factors were considered for each surveillance system component: 1) the number of wild birds tested; 2) whether the tested wild birds were considered “higher risk species” (HRS) for being infected with AI; 3) the location of the birds tested with respect to counties designated as a priority for surveillance; and 4) the probability that the birds tested might yield a positive AI virus result based on surveillance results in wild birds across Europe. The number of birds tested by both surveillance types was greatly reduced after the strategy change. The proportion of birds sampled in priority counties also significantly decreased in the second year for both active and passive surveillance. However, the proportion of HRS sampled by active surveillance significantly increased, while a significant decrease in these species was seen for passive surveillance in the second year. The derived probability scores for detecting AI based on European surveillance results indicated a reduction in sensitivity for H5N1 highly pathogenic AI detection by passive surveillance. The methods developed to evaluate AIWBS in GB may be applicable to other European Union countries. The results also reflect the complex issues associated with evaluation of disease surveillance in wildlife populations in which the disease ecology is only partially understood.

In Denmark and Greenland, extensive surveillance of avian influenza (AI) viruses in wild bird populations has been conducted from 2007 through 2010. In Denmark, the surveillance consisted of passive surveillance of wild birds found dead or sick across Denmark and active surveillance of apparently healthy live birds in waterfowl reservoirs and along migratory flyways, birds living in proximity to domestic poultry, and hunted game birds. Dead birds were sampled by oropharyngeal swabbing. Healthy live wild birds were captured with nets, traps, or by hand and were sampled by swabbing of the oropharyngeal and cloacal tracts, or swabs were collected from fresh fecal droppings. Hunted game birds were delivered to game-handling establishments, where each bird was sampled by oropharyngeal and cloacal swabbing. During the 2007–10 period, a total of 11,055 wild birds were sampled in Denmark, of which 396 were birds that were found dead. In Greenland, samples were collected mainly from fecal droppings in breeding areas. Samples from 3555 live and apparently healthy wild birds were tested. All swab samples were tested by pan-influenza reverse transcriptase–PCR (RT-PCR), and the positive samples were further tested by H5/H7 specific RT-PCRs. H5/H7-positive samples were subjected to hemagglutination cleavage site sequencing for pathotyping. In addition, all RT-PCR–positive samples were subjected to virus isolation, and the virus isolates were subsequently subtyped. In Denmark, low pathogenic (LP) H5 viruses were detected throughout the period, in addition to a few LPAI H7 and several other subtypes. In Greenland, very few samples were positive for AI. None of them were found to be of the H5 or H7 subtypes by RT-PCR. Isolation of these viruses in eggs was unsuccessful; thus, they were not subtyped further. The findings did, however, demonstrate the presence of LPAI viruses in Greenland. For several water bird species overwintering in North America and northwest Europe, respectively, Greenland constitutes a common breeding area. This raises the possibility that viruses could be transmitted to North America via Greenland and vice versa. In Denmark, the screenings for AI showed LPAI viruses to be naturally occurring in the wild bird population, particularly in waterfowl. The occurrence of AI viruses in the wild bird population may pose a risk for AI infections in Danish poultry.

Within the framework of the surveillance program for the early detection of H5 and H7 subtypes of avian influenza (AI) viruses, samples from 2547 wild birds of different species that were collected between 2006 and 2010 were examined by PCR-based methods. AI viruses of various subtypes were detected in 4.4% of birds from four different orders: Anseriformes, Ciconiiformes, Charadriiformes, and Pelecaniformes. Highly pathogenic avian influenza (HPAI) H5N1 viruses were detected only in 2006. HPAI H5N1 virus was confirmed in 1.9% of birds from four different species. Comparison of nucleotide sequences of the H5N1 hemagglutinin gene indicated that two different HPAI H5N1 viruses from the European–Middle Eastern–African clade 1 had been introduced into Slovenia, despite the relatively short duration of the HPAI outbreak. Low pathogenic avian influenza (LPAI) viruses were detected in 2.5% of birds during a 5-yr period. The subtypes H1, H2, H3, H4, H5, H7N7, H8, H10, H11, and H13N6 were determined in 18 out of 64 cases. The highest prevalence (81%) of LPAI viruses, including the H5 subtype, were found in birds sampled as a part of the “active” surveillance system.

Wild waterfowl are considered the natural reservoir of type A influenza viruses, and the migratory nature of many waterfowl species presents a possible vehicle for global dissemination of these infectious agents. In order to fully understand the ecology of influenza viruses, multiyear surveillance efforts are critical, particularly in understudied areas, such as waterfowl wintering areas. Herein we report results obtained during the fifth year of a 5-yr avian influenza virus (AIV) surveillance project conducted on waterfowl wintering grounds of the Texas Coast. During year 5, the 2009–2010 hunting season (September, November–January), 655 cloacal swabs were collected from hunter-harvested waterfowl and screened for AIV by real-time RT-PCR (rRT-PCR) followed by virus isolation on all positive samples. Molecular methods were used for subtyping all AIV isolates. Sixty-five (9.5%) samples were positive for AIV by rRT-PCR, and 24 (3.7%) AIVs were isolated. Eight different hemagglutinin (H3, 4, 5, 6, 8, 9, 10, and 11) and seven different neuraminidase (N1, 2, 3, 4, 6, 8, and 9) subtypes were identified. This was the first year H8 and H9 were isolated throughout the 5-yr survey. Our results support the fact that continued multiyear surveillance of natural reservoirs, particularly in understudied areas, is needed in order to better understand the ecology of AIVs in nature.

The Azov and Black Sea basins are part of the transcontinental wild bird migration routes from Northern Asia and Europe to the Mediterranean, Africa, and Southwest Asia. These regions constitute an area of transit, stops during migration, and nesting for many different bird species. From September 2010 to September 2011, a wild bird surveillance study was conducted in these regions to identify avian influenza viruses. Biological samples consisting of cloacal and tracheal swabs and fecal samples were collected from wild birds of different ecological groups, including waterfowl and sea- and land-based birds, in places of mass bird accumulations in Sivash Bay and the Utlyuksky and Molochniy estuaries. The sampling covered the following wild bird biological cycles: autumn migration, wintering, spring migration, nesting, and postnesting seasons. A total of 3634 samples were collected from 66 different species of birds. During the autumn migration, 19 hemagglutinating viruses were isolated, 14 of which were identified as low pathogenicity avian influenza (LPAI) virus subtypes H1N?, H3N8, H5N2, H7N?, H8N4, H10N7, and H11N8. From the wintering samples, 45 hemagglutinating viruses were isolated, 36 of which were identified as LPAI virus subtypes H1N1, H1N? H1N2, H4N?, H6N1, H7N3, H7N6, H7N7, H8N2, H9N2, H10N7, H10N4, H11N2, H12N2, and H15N7. Only three viruses were isolated during the spring migration, nesting, and postnesting seasons (serotypes H6, H13, and H16). The HA and NA genes were sequenced from the isolated H5 and N1 viruses, and the phylogenetic analysis revealed possible ecological connections between the Azov and Black Sea regions and Europe. The LPAI viruses were isolated mostly from mallard ducks, but also from shellducks, shovelers, teals, and white-fronted geese. The rest of the 14 hemagglutinating viruses isolated were identified as different serotypes of avian paramyxoviruses (APMV-1, APMV-4, APMV-6, and APMV-7). This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.

In the present study, fecal samples obtained from kelp gulls (Larus dominicanus), brown-hooded gulls (Larus maculipennis), and Olrog's gulls (Larus atlanticus) on the coast of the District of Pinamar, and grey-hooded gulls (Larus cirrocephalus) on the coast of the Lagoon Salada Grande and surrounding wetlands, General Madariaga, Buenos Aires Province, Argentina, were tested for evidence of avian influenza virus over a period of 3 yr. This surveillance in free-living wild birds in the Buenos Aires Province started in October 2008. Additional samples, which included cloacal swabs, tracheal swabs, or pooled organs, were obtained from sick or dead gulls that arrived at the Fundación Ecológica Pinamar or were provided by the Dirección de Seguridad en Playas, Municipalidad de Pinamar. Samples were pooled according to date, species, and area. Pooled samples were inoculated in 9- to 11-day-old eggs, and after 5 days, allantoic fluids were tested for evidence of hemagglutination. None of the samples was positive for avian influenza viruses.

In the Veneto region (northern Italy), some geographic areas in the Po Valley have a large concentration of industrial poultry farms and are located close to wet areas with high populations of wild waterfowl. Live decoy birds belonging to the orders of Anseriformes and Charadriiformes can constitute a “bridge” for avian influenza (AI) viruses between the wild reservoir and the rural holdings where live decoy birds are usually kept, sometimes together with poultry. Thus, the use of live decoy birds during bird hunting could increase the risk of exposure of poultry farms to AI viruses. Since 2008, this kind of hunting has been strictly regulated with regard to the detection and use of live decoy birds. In order to guarantee the application of appropriate AI risk-modulating and monitoring measures in the management of the live decoys according to the European Union (EU) provisions, a solid and well-structured information system has been created. The Regional Data Bank (RDB) of farms and livestock, which has been operating since 1997, also contains data on farms and poultry movements. Therefore, the RDB management software was updated to collect data from the hunters who keep live decoy birds, and specific functions were integrated to ensure the traceability of these birds. Each live decoy bird has been identified by an irremovable ring. The individual code of each ring is recorded in the RDB and linked to both the holder's code and the hunting area. Transfers and death/slaughtering of the registered birds are recorded, too. The activation of a computerized data collection system has proven to be a prerequisite for the implementation of a control system for live decoy birds and provides an essential tool for the management of AI emergencies.

Avian influenza virus (AIV) surveillance has been scarce in most countries of Latin America and the Caribbean. Historically, avian influenza surveillance efforts in Central and South America have been localized in places where outbreaks in poultry have occurred. Since the emergence of the H5N1 subtype in Asia, active surveillance in wild birds has increased in a number of Latin American countries, including Barbados, Guatemala, Argentina, Brazil, Mexico, and Peru. A broad diversity of virus subtypes has been detected; however, nucleotide sequence data are still limited in comparison to other regions of the world. Here we review the current knowledge of AIV in Latin America, including phylogenetic relationships among publicly available viral genomes. Overall AIV reports are sparse across the region and the cocirculation of two distinct genetic lineages is puzzling. Phylogenetic analysis reflects bias in time and location where sampling has been conducted. Increased surveillance is needed to address the major determinants for AIV ecology, evolution, and transmission in the region.

Sardinia is a Mediterranean island with a long geological history, leading to a separation process from continental Europe during the Miocene. As a consequence, in this insular habitat some wild bird species developed endemic forms, some of which are currently threatened. The aim of this study is to evaluate the possible animal health risk associated with a potential avian influenza virus (AIV) circulation in Sardinian wild bird populations. Overall, 147 cloacal swabs were sampled in the Sardinia region from June 2009 to September 2011. Samples were obtained from 12 taxonomic orders, including 16 families and 40 species of birds. Based on the endangered host status or on the ecology of the host-virus interaction, samples were categorized into three groups of species: 1) endemic, endangered, or both (17 species); 2) potential reservoir (21 species); and 3) potential spillover (two species). Cloacal swabs were tested by reverse transcription (RT)-PCR for influenza A virus matrix gene amplification. Forty-one serum samples were tested by nucleoprotein-enzyme-linked immunosorbent assay (NP-ELISA) for antibodies against influenza A virus nucleoprotein and by hemagglutination inhibition assay for detection of seropositivity against H5 and H7 AIV subtypes. No cloacal swabs tested RT-PCR positive for AIV, whereas two weak seropositive results were detected by NP-ELISA in a mallard (Anas platyrhynchos) and in a yellow-legged gull (Larus michahellis). The low or absent AIV circulation detected in Sardinia's wild birds during the study suggests a naїve status in these avian populations. These data provide new information on AIV circulation in Sardinia's wild birds that could be applied to implement conservation strategies for threatened species.

The objective of this study was to demonstrate the effects of the nature of the information collected through passive surveillance on the detection of space-time clusters of highly pathogenic avian influenza virus (HPAIV) H5N1 cases reported among dead wild birds in Denmark and Sweden in 2006. Data included 1469 records (109 cases, 1360 controls) collected during the regional epidemic between February and June by passive surveillance of dead wild birds. Laboratory diagnoses were obtained by PCR methods and/or virus isolation. The nature of available information influences both the type of model suitable for analysis and its parameterization. Here, we explored four alternative scan-based methods, suitable for detection of clusters only when case data (univariate permutation model), case and hypothesized epidemiological variables (multivariate permutation model), case and control data (univariate Bernoulli model), and case, control, and hypothesized epidemiological variables (multivariate Bernoulli model) are available. Tufted ducks were particularly common among infected wild bird species detected in Denmark and Sweden during the initial phases of this epidemic, and species group (tufted ducks [62 cases, 57 controls] vs. other wild bird species [47 cases, 1303 controls]) was considered in the multivariate models as a covariate potentially associated with clustering. Bernoulli and permutation scan analyses both detected multiple significant (P < 0.01) clusters with similar locations, but with certain differences in their numbers and sizes. The observed-to-expected case ratios in the two clusters detected by the multivariate Bernoulli scan model were substantially heterogeneous. However, the permutation model detected only one of the Swedish clusters and only pinpointed the heterogeneity between species on clustering in the same Danish cluster as detected by the Bernoulli model. The output of the methods described here were shown to be highly sensitive to the choice of the probability model for cases and the choice of plausible assumptions to parameterize the scan statistic tests. The results of the multivariate Bernoulli suggest that with noncase information regarding a potential risk factor, such as species of birds, this method is sensitive and efficient in identifying high-risk areas and time periods for regional occurrence of HPAIV and potentially for similar infectious diseases. Results here demonstrate the impact that the nature of the collected information has on the epidemiological investigation of outbreaks. Results show the importance of collecting information on control data and on variables hypothesized to influence disease risk on the identification of periods of time and locations at high risk for the disease and risk factors associated with clustering as part of the national and international surveillance systems.

A highly pathogenic avian influenza (HPAI) outbreak in the United States will initiate a federal emergency response effort that will consist of disease control and eradication efforts, including quarantine and movement control measures. These movement control measures will not only apply to live animals but also to animal products. However, with current egg industry “just-in-time” production practices, limited storage is available to hold eggs. As a result, stop movement orders can have significant unintended negative consequences, including severe disruptions to the food supply chain. Because stakeholders' perceptions of risk vary, waiting to initiate communication efforts until an HPAI event occurs can hinder disease control efforts, including the willingness of producers to comply with the response, and also can affect consumers' demand for the product. A public-private-academic partnership was formed to assess actual risks involved in the movement of egg industry products during an HPAI event through product specific, proactive risk assessments. The risk analysis process engaged a broad representation of stakeholders and promoted effective risk management and communication strategies before an HPAI outbreak event. This multidisciplinary team used the risk assessments in the development of the United States Department of Agriculture, Highly Pathogenic Avian Influenza Secure Egg Supply Plan, a comprehensive response plan that strives to maintain continuity of business. The collaborative approach that was used demonstrates how a proactive risk communication strategy that involves many different stakeholders can be valuable in the development of a foreign animal disease response plan and build working relationships, trust, and understanding.

The perception of risks of exposure to avian influenza and other poultry diseases among adults in Tanzania is influenced by their previous experiences, beliefs, and values, which can stand in the way of learning new approaches. We tested a novel disease risk communication approach centered on elementary school pupils, involving their teachers and parents. Age-appropriate training modules were developed and taught to teachers who then taught their pupils through extracurricular activities. The pupils practiced what they learned through club projects and subsequently transmitted what they learned to their parents. In 2009 we developed a poultry health and production curriculum as part of efforts to prevent and control poultry diseases, including avian influenza, in Tanzania. The curriculum developed for veterinarians and veterinary paraprofessionals was adapted for use with elementary school children and translated into Kiswahili. Twenty teachers from 10 primary schools in Mzumbe ward, Morogoro, were trained by poultry veterinary extension experts on teaching the curriculum to standard 5 pupils (ages 11–12 yr). Pupils and teachers practiced the curriculum in four demonstration chicken coops established on the grounds of the Changarawe, Lubungo, Masanze, and Mzumbe primary schools. By October 2011, at the conclusion of the funded project, a total of 202 girls and 193 boys had been trained. Additionally, 34 adults from surrounding villages made official learning tours to the schools and received training from their children and teachers involved in the projects. With at least 75% of the 395 pupils coming from different households, it can be safely assumed that over 250 households have heard about poultry disease risks and how to manage poultry to prevent those risks.

Influenza pandemics pose a continuous risk to human and animal health and may engender food security issues worldwide. As novel influenza A virus infections in humans are identified, pandemic preparedness strategies necessarily involve decisions regarding which viruses should be included for further studies and mitigation efforts. Resource and time limitations dictate that viruses determined to pose the greatest risk to public or animal health should be selected for further research to fill information gaps and, potentially, for development of vaccine candidates that could be put in libraries, manufactured and stockpiled, or even administered to protect susceptible populations of animals or people. A need exists to apply an objective, science-based risk assessment to the process of evaluating influenza viruses. During the past year, the Centers for Disease Control and Prevention began developing a tool to evaluate influenza A viruses that are not circulating in the human population but pose a pandemic risk. The objective is to offer a standardized set of considerations to be applied when evaluating prepandemic viruses. The tool under consideration is a simple, additive model, based on multiattribute decision analysis. The model includes elements that address the properties of the virus itself and population attributes, considers both veterinary and human findings, and integrates both laboratory and field observations. Additionally, each element is assigned a weight such that all elements are not considered of equal importance within the model.

We report the first occurrence of pandemic (H1N1) 2009 virus [A(H1N1)pdm09] infection on two epidemiologically linked turkey breeder premises in the United Kingdom during December 2010 and January 2011. Clinically, the birds showed only mild signs of disease, with the major presenting sign being an acute and marked reduction in egg production, leading to the prompt reporting of suspected avian notifiable disease for official investigation. Presence of A(H1N1)pdm09 infection in the United Kingdom turkey breeder flocks was confirmed by detailed laboratory investigations including virus isolation in embryonated specific pathogen-free fowls' eggs, two validated real-time reverse transcription-PCR tests, and nucleotide sequencing of the hemagglutinin and neuraminidase genes. These investigations revealed high nucleotide identity with currently circulating human A(H1N1)pdm09 strains, suggesting that human-to-poultry transmission (reverse zoonosis) was the most likely route of infection. Peak levels of human influenza-like illness community transmission also coincided with the onset of clinical signs in both affected turkey breeder flocks. This case demonstrated the value of the existing passive surveillance framework and associated veterinary and laboratory infrastructure that enables the detection and management of both exotic and new and emerging disease hazards and risks. The case also presents further evidence of the susceptibility of turkeys to infection with influenza A viruses of nonavian origin.

Highly pathogenic (HP) and low pathogenic (LP) avian influenza viruses (AIVs) belonging to H5 and H7 subtypes have been found to be associated with human infection as the result of direct transmission from infected poultry. Human infections by AIVs can cause mild or subclinical disease, and serosurveys are believed to represent an important tool to identify risk of zoonotic transmission. Therefore, we sought to examine Italian poultry workers exposed during LPAI and HPAI outbreaks with the aim of assessing serologic evidence of infection with H5 and H7 AIVs. From December 2008 to June 2010 serum samples were collected from 188 poultry workers and 379 nonexposed controls in Northern Italy. The hemagglutination inhibition (HI) assay using horse red blood cells (RBCs) and a microneutralization (MN)–enzyme-linked immunosorbent assay test were used to analyze human sera for antibodies against the following H5 and H7 LPAI viruses: A/Dk/It/4445/07(H5N2); A/Ty/It/2369/09(H5N7); A/Ty/It/218-193/10; A/Ck/It/3775/99(H7N1); A/Ty/It/214845/03(H7N3); and A/Dk/It/332145/09(H7N3). Since previous studies identified low antibody titer to AIVs in people exposed to infected poultry, a cutoff titer of ≥1∶10 was chosen for both serologic assays. Only HI-positive results confirmed by MN assay were considered positive for presence of specific antibodies. The Fisher exact test was used to analyze differences in seroprevalence between poultry workers and control groups, with the significance level set at P < 0.05. MN results showed a proportion of H7-seropositive poultry workers (6/188, i.e., 3.2%), significantly higher than that of controls (0/379), whereas no MN-positive result was obtained against three H5 LPAI subtypes recently identified in Italy. In conclusion, the survey indicated that assessing seroprevalence can be an important tool in risk assessment and health surveillance of poultry workers.